Among the risks inherent in handling or being transfused with blood, blood proteins, or other blood components is the risk of infection from pathogenic contaminants, including human immunodeficiency viruses (HIV) and hepatitis viruses. Virucidal methods, including heat, solvent-detergent, and gamma irradiation have been used to produce non-infectious plasma derivatives, but such methods are ineffective or are too harsh to be used for decontamination of whole blood, red cells or platelets. Any treatment that damages or introduces harmful or undesirable contaminants into the product is unsuitable to decontaminate a product intended for transfusion.
Because of the critical need for transfusible red blood cells, it is of great importance to develop methods that can be readily used to decontaminate cellular blood components and whole blood without substantially or irreversibly altering or harming them.
At least 75% of the red cells that are transfused must be circulating 24 hours after their transfusion. The shelf-life and suitability of red blood cells for transfusion is determined on this basis. The concentrations of ATP and of 2,3 diphosphoglycerate (2,3 DPG) and the morphology of red cells serve as indicators of the suitability of such cells for transfusion. During prolonged storage or as a result of harsh treatments, human red blood cells undergo changes that include decreases in the cellular levels of ATP and 2,3 DPG and changes in cellular morphology. For example, during storage, the concentration of ATP, after a brief initial rise, progressively declines to about 50% of its initial level. The fluidity of the cell membranes of red cells, which is essential for the passage of such cells through the narrow channels in the spleen and liver, is correlated with the level of ATP. As the level of ATP declines, the fluidity of the cellular membrane decreases rendering the cells unsuitable for transfusion. The level of 2,3 DPG falls rapidly after about 3 or 4 days of storage and approaches zero after about 10 days. 2,3 DPG is associated with the ability of the hemoglobin in the red cells to deliver oxygen to the tissues.
Solutions that prolong the shelf life of red cells are known (Meryman et al., U.S. Pat. No. 4,585,735 (incorporated herein by reference)). Typically such solutions contain citrate, phosphate, glucose, adenine, and other ingredients and function to prolong shelf life by maintaining the levels of ATP and 2,3 DPG in the cells. Solutions that contain a penetrating salt, such as ammonium acetate, in addition to phosphate, glucose, and adenine, and that are hypotonic with respect to molecules that are unable to penetrate the cell membrane, have been shown to maintain the levels of ATP for more than 100 days of refrigeration (Meryman et al., supra).
Decontamination treatments that inactivate contaminating pathogens, but that do not harm the cellular fractions of blood are not readily available. Decontamination procedures presently include the use of photosensitizers, which, in the presence of oxygen and upon exposure to light, including wavelengths absorbed by the photosensitizer, inactivate viruses (EP 0 196 515, published 08.10.86, to Baxter Travenol Laboratories, Inc.). Typically such photochemicals are dyes or other compounds that readily absorb UV or visible light in the presence of oxygen. Such compounds include psoralen derivatives (U.S. Pat. No. 4,748,120 to Wiesehahn), porphyrin derivatives (U.S. Pat. No. 4,878,891 to Judy et al.) and other photosensitizers. Often, however, such treatment also damages cellular blood components.
The virucidal activity of these compounds is realized when the absorption spectrum of the photosensitizer does not significantly overlap the absorption spectra of pigments present in the blood. In order to minimize cellular damage, it is advantageous if the photosensitizer selectively binds to a component of the virus that is not present in red cells or platelets or, if present therein, that is not essential to red cells' or platelets' function, and is not toxic to these cells. It is also preferable if the photodynamic treatment inactivates extracellular and intracellular virus as well as cells containing provirus. It is beneficial if the virucidal activity of the photosensitizer is not inhibited by the presence of plasma proteins.
Photochemicals such as the psoralens (U.S. Pat. No. 4,748,120 to Wiesehahn) damage nucleic acids in the presence of light while the porphyrins (U.S. Pat. No. 4,878,891 to Judy et al.) and merocyanine 540 (MC 540), (U.S. Pat. No. 4,775,625 to Sieber) cause membrane damage in the presence of light and oxygen and thereby inactivate viruses and bacteriophages.
Among the problems that occur during decontamination with psoralen and porphyrin derivatives is that they apparently bind to blood components, such as albumin (Prodouz, Transfusion 29:42S (1989)). Prodouz studied the effect of MC 540 on platelets and the influence of albumin on MC 540's virucidal activity. Platelets exhibited a MC dose-dependent decrease in response to thrombin in the absence of light. In the presence of light and MC 540, the platelets aggregated. Albumin prevented that aggregation and inhibited the inactivation of viral contaminants by MC 540 plus light.
Similarly, because of such competitive inhibition reactions with blood or plasma components, other dyes have not been suitable for decontaminating blood, cellular blood components, or any blood derived products containing high plasma concentrations. As the plasma concentration increases, the percentage of viral inactivation substantially decreases.
The phenothiazin-5-ium dyes, which include methylene blue, toluidine blue O, thionine, azure A, azure B, and azure C, are useful for inactivating animal viruses (Swartz, U.S. Pat. Nos. 4,407,282, 4,402,318, 4,305,390, and 4,181,128). These dyes, however, have not been used to inactivate pathogens in whole blood or in cellular blood components because red cells readily take up or bind such dyes (Sass et al., J. Lab. Clin. Med. 73:744-752 (1969)). Methylene blue and visible light damage guanine residues of nucleic acids (Simon et al., J. Mol. Biol. 4:488-499 (1962)). Methylene blue and white light produce 8-hydroxyguanine in DNA (Floyd et at., Arch. Biochim. Biophys. 273:106-111 (1989)). In addition, Girotti demonstrated that photosensitized oxidation of biological membranes is deleterious to membrane structure and function and showed that methylene blue cross-links the membrane protein, spectrin, in erythrocytes exposed to visible light and oxygen (Girotti, Biochim. Biophys. Acta. 602:45-56 (1980)). Thus, because of these and other potentially deleterious effects, phenothiazin-5-ium dyes have not been selected as photosensitizers for decontaminating blood or cellular blood components.
No method has proven fully successful for decontaminating whole blood, cellular blood components during storage, or compositions containing concentrated blood components, including high levels of plasma. Because of the AIDS epidemic there is, however, an acute need to develop a safe method whereby pathogenic contaminants, particularly HIV and hepatitis, in blood or in cellular blood components can be inactivated without rendering the blood or cellular blood components unsuitable for transfusion.